卵巣癌の制癌剤抵抗性に関与するアポトーシスシグナルの分子生物学的解析-遺伝子治療の分子標的の確立をめざして-

卵巢癌抗癌药物耐药性中涉及的细胞凋亡信号的分子生物学分析 - 旨在建立基因治疗的分子靶标 -

• 批准号: 11671613
• 负责人: MANDAI Masaki
• 金额: $ 2.3万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671613/
• 关键词: drug resistance; apoptosis; gene therapy; ovarian cancer; cisplatin; bax; adenovirus; paclitaxel; IGF-1; 制癌制抵抗性

The objective of this study is to clarify the association between the intracellular apoptotic signaling and the cytotoxic/therapeutic effect of cisplatin in ovarian cancer. Then, according to the findings obtained, we tried to develop a new strategy to manage cisplatin-resistant ovarian cancer.Firstly, the influence of apoptosis on the effect of cisplatin is examined. Treatment with cisplatin in cisplatin-sensitive ovarian cancer cell line A2780 markedly induced apoptosis, which was detected both by morphological examination as well as quantification of apoptosis using cell death ELISA method or measurement of caspase 3 activity. In contrast, cisplatin in the same dose did not induce significant apoptosis in cisplatin-resistant SK-OV-3 cell line. In addition, an inhibitor of caspase 3, Ac-DEVD-CHO, reduced the cytotoxity of cisplatin, suggesting that the effect of cisplatin is closely linked to the induction of apoptosis in ovarian cancer cells.Secondly, the role of pro-apoptotic Bax p … More rotein in the development of cisplatin-resistance of ovarian cancer was investigated. Immunohistochemical analysis revealed that decreased expression of Bax protein correlated with poor prognosis of patient with ovarian cancer. In vitro, Bax expression was attenuated in cisplatin-resistant ovarian cancers, A2780/cDDP and SK-OV-3, compared with cisplatin sensitive A2780. These findings suggest that the disturbance in pro-apoptotic signaling through Bax play an inportant role in the development of cisplatin-resistance. Accordingly, we tried to induce Bax protein in ovarian cancer cells using recombinant adenovirus which highly express the Bax protein. The pro-apoptotic effect of Bax induction was similarly intensive either in A2780 or A2780/cDDP, while it was minimum in SK-OV-3. Preliminarily, adenovirus-mediated transfer of the Bax gene into human ovarian xenografts showed suppressive effect on tumor growth. Therefore, adenovirus-mediated introduction of Bax may be a useful tool to manage a part of cisplatin-resistant ovarian cancer.Thirdly, the influence of gonadotropins, luteinizing hormone (LH) and human chorionic gonadotropin (hCG), on the apoptosis of ovarian cancer was examined. More than a half of ovarian cancer specimens expressed LH/hCG receptor. hCG treatment markedly reduced cisplatin-induced apoptosis in LH/hCG receptor-positive ovarian cancer cell line, OVCAR-3, and induced insulin-like growth factor 1 (IGF-1 ) expression. IGF-1 also inhibited cisplatin-induced apoptosis and a neutralizing antibody to IGF-1 receptor restored apoptosis in OVCAR-3, suggesting that IGF-1 mediates anti-apoptotic signaling from hCG.Wortmannin, an inhibitor of phosphatidyl inositol 3 kinase (PI3K), blocked the anti-apoptotic effect of IGF-1 to restore the cisplatin-induced apoptosis. This indicates that specific phosphorylation signal is related to cisplatin-sensitivity/resistance of ovarian cancer.In conclusion, both the intrinsic disturbance of intracellular apoptosis signal including Bax and the presence of anti-apoptotic extrinsic factors such as IGF-1 may cause the resistance to chemotherapy in ovarian cancer. Consequently, to regulate apoptosis-related gene and to facilitate apoptosis by the methods including adenovirus-mediated gene therapy may be a hopeful strategy to overcome drug-resistance. Less

本研究的目的是阐明细胞内凋亡信号与顺铂在卵巢癌中的细胞毒性/治疗作用之间的关系。然后，根据所获得的结果，我们试图开发一种新的策略来管理顺铂耐药的卵巢癌。首先，研究细胞凋亡对顺铂疗效的影响。顺铂处理对顺铂敏感的卵巢癌细胞系A2780显著诱导凋亡，这是通过形态学检查以及使用细胞死亡ELISA方法或测量caspase 3活性来定量凋亡来检测的。而相同剂量的顺铂对耐药细胞株SK-OV-3无明显的凋亡诱导作用。此外，caspase 3抑制剂Ac-DEVD-CHO可降低顺铂的细胞毒性，提示顺铂的作用与诱导卵巢癌细胞凋亡密切相关。 ...更多信息 研究了鱼藤素在卵巢癌顺铂耐药发展中的作用。免疫组化分析显示Bax蛋白表达降低与卵巢癌患者预后不良相关。在体外实验中，与顺铂敏感的A2780相比，Bax在顺铂耐药的卵巢癌A2780/cDDP和SK-OV-3中表达减弱。这些结果表明，通过Bax的促凋亡信号的干扰发挥了重要作用的顺铂耐药的发展。因此，我们尝试使用高表达Bax蛋白的重组腺病毒在卵巢癌细胞中诱导Bax蛋白。Bax诱导的促凋亡作用在A2780或A2780/cDDP中类似地强烈，而在SK-OV-3中最小。腺病毒介导的Bax基因转移到人卵巢移植瘤中对肿瘤生长有抑制作用。因此，腺病毒介导的Bax基因的导入可能是治疗部分顺铂耐药卵巢癌的有效工具。第三，研究促性腺激素促黄体生成素（LH）和人绒毛膜促性腺激素（hCG）对卵巢癌细胞凋亡的影响。一半以上的卵巢癌标本表达LH/hCG受体。hCG处理显著减少顺铂诱导的LH/hCG受体阳性卵巢癌细胞系OVCAR-3的凋亡，并诱导胰岛素样生长因子1（IGF-1）的表达。IGF-1还抑制顺铂诱导的OVCAR-3细胞凋亡，IGF-1受体中和抗体可恢复顺铂诱导的OVCAR-3细胞凋亡，表明IGF-1介导hCG的抗凋亡信号，磷脂酰肌醇3激酶（PI 3 K）抑制剂Wortmannin可阻断IGF-1的抗凋亡作用，恢复顺铂诱导的凋亡。提示卵巢癌化疗耐药与特异性磷酸化信号有关，卵巢癌化疗耐药的机制可能与细胞内凋亡信号（Bax）的内在紊乱和抗凋亡的外源性因子（IGF-1）的存在有关。因此，利用腺病毒等基因治疗手段调控肿瘤相关基因，促进肿瘤细胞凋亡，可能是克服耐药的一个有希望的策略。少

项目成果

Masaki MANDAI: "A Novel Approach to Modurate the Expression of Apoptosis-related Genes as a Treatment Strategy of Chemo-resistant Ovarian Cancer"ACTA OBST GYNAEC JPN. Vol.51. 575-585 (1999)

Masaki MANDAI：“调节细胞凋亡相关基因表达的新方法作为化疗耐药性卵巢癌的治疗策略”ACTA OBST GYNAEC JPN。

鶴田優子: "婦人科癌の浸潤・転移とその臨床「卵巣癌の転移にかかわる遺伝子」"産婦人科の実際. 49(6). 765-774 (2000)

Yuko Tsuruta：“妇科癌症的侵袭和转移及其临床实践``与卵巢癌转移相关的基因。妇产科实践。49（6）。765-774（2000）

万代昌紀: "卵巣癌細胞のアポトーシス抑制に関与する遺伝子群を標的とした新しい癌化学療法の基礎的研究"日本産科婦人科学会雑誌. 51. 575-585 (1999)

Masanori Bandai：“针对参与抑制卵巢癌细胞凋亡的基因的新型癌症化疗的基础研究”日本妇产科学会杂志 51. 575-585 (1999)。

万代昌紀: "がん治療のあゆみ 第19巻"(財)がん集学的治療研究財団. 8 (2000)

万代正德：《癌症治疗史第19卷》癌症多学科治疗研究基金会8（2000）。

鶴田優子: "Combination effect of adenovirus-mediated pro-apoptotic Bax gene transfer with cisplatin or paclitaxel treatment in ovarian cancer cell lines."European Journal of Cancer. 37. 531-541 (2001)

Yuko Tsuruta：“腺病毒介导的促凋亡 Bax 基因转移与顺铂或紫杉醇治疗在卵巢癌细胞系中的组合效应。”欧洲癌症杂志 37. 531-541 (2001)。